M2D4: Complete Western / Damage DNA3/20/14

1. Pre-lab discussion: Review WB & RE Digest

2. Set-up RE Digest

3. 2nd step of WB

4. Gel purify cut DNA for NHEJ assay!

5. Spring Break!

Validate the system - confirm decrease in Ku80 expression .

250

150

10075

503725

15

MW (kDa)

CHO-K1 cells CHO-xrs6 cells

Western blot detection systems:

Chemical reaction pathway from sigma.com: http://www.sigmaaldrich.com/technical-documents/articles/biofiles/colorimetric-alkaline.html

NBT - substrate B

BCIP - substrate A

Colorimetric:

Chemiluminescent: Fluorescent:

Today you will prepare (and validate) your damaged DNA:

20.109-

1. Set-up digest — Dilute enzyme in: 2. Finish WB — handle with care 3. Gel purify digest product — Leave a space!

Today in Lab:

1. Microbiota re-write (final due on M2D6) 2. . 3. . 4. .

Due after spring break (woo hoo!):